Water soluble polymer surfactant for synthesis of functionalized polystyrene nanobeads towards detection of bilirubin in human serum

Abstract

The present invention provides a water soluble polymer surfactant (PS-DGlu) of formula I which is utilized for synthesis of functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) which in turn is useful for the detection of bilirubin in human serum. The present invention further provides a process for the preparation of the water soluble polymer surfactant (PS-DGlu) of formula (I) and a mini emulsion polymerization process for the synthesis of PSG-OPV-n. wherein, n is 30-50. The PSG-OPV-n nanosensor beads show selectivity towards detection of bilirubin in presence of interferences such as glucose, sucrose, metal ions, cholesterol, and biliverdin with limit of detection of 20 nM. Ultimately, the invention also provides a kit for visual detection of bilirubin in human serum.

We claim: 
     
       1. A water soluble polymer surfactant (PS-DGlu) of formula (I) comprising a hydrophobic polystyrene and a hydrophilic glucuronic acid units joined together with a triazole moiety; 
       
         
           
           
               
               
           
         
         wherein n is 30-50. 
       
     
     
       2. A process for the preparation of the water soluble polymer surfactant of  claim 1 , comprising the steps of:
 a) adding an iodine solution to a mixture of D-glucuronic acid and acetic anhydride at a temperature in the range of 0° C. to −5° C. to obtain a reaction mixture; stirring the mixture at a temperature in the range of 0° C. to −5° C. for a time period ranging from 2 to 3 hours, followed by stirring at a temperature in the range of 25° C. to 30° C. for a time period ranging from 2.5 to 4 hours to obtain penta-acetate glucuronic acid (1) 
 
       
         
           
           
               
               
           
         
         b) refluxing the penta-acetate glucuronic acid (1) as obtained in step (a) in dry methanol at a temperature in the range of 60° C. to 90° C. for a time period ranging from 12 to 26 hours to obtain 1, 2, 3, 4-Tetra-O-acetyl-methyl-β-D-glucuronide (2) 
       
       
         
           
           
               
               
           
         
         c) adding TMS-azide and tin (IV) chloride to the 1, 2, 3, 4-Tetra-O-Acetyl-methyl-β-D-Glucuronide (2) as obtained in step (b) in a first solvent, followed by stirring at a temperature in the range of 25 to 30° C. for a time period ranging from 15 to 20 hours to obtain 2, 3, 4-tri-O-acetyl-1-azido-1-deoxy-β-D-glucuronic acid methyl ester (3) 
       
       
         
           
           
               
               
           
         
         d) heating a reaction mixture of 4-bromostyrene, ethynyltrimethylsilane, copper(I) iodide and triethyl amine at a temperature in the range of 50 to 60° C. for a time period ranging from 5 to 15 minutes, followed by adding bis(triphenylphosphine) palladium (II) dichloride followed by stirring at a temperature in the range of 50 to 60° C. for a time period ranging from 16 to 17 hours to obtain 4-(trimethylsilane)ethynylstyrene (4) 
       
       
         
           
           
               
               
           
         
         e) adding tetra-n-butyl ammonium fluoride to the 4-(Trimethylsilane)ethynylstyrene (4) as obtained in step (d) in a second solvent to obtain a reaction mixture, followed by stirring the reaction mixture at a temperature in the range of 25 to 30° C. for a time period ranging from 1 to 2 hours to obtain 4-ethynylstyrene (5) 
       
       
         
           
           
               
               
           
         
         f) adding a copper sulfate and sodium ascorbate in a water to the 4-ethynylstyrene (5) as obtained in step (e) and the 2, 3, 4-tri-O-acetyl-1-azido-1-deoxy-β-D-glucuronic acid methyl ester (3) as obtained in step (c) in a third solvent to obtain a reaction mixture; stirring the reaction mixture at a temperature in the range of 25° C. to 30° C. for a time period ranging from 24 to 25 hours to obtain (2R, 3S, 4R, 5S, 6R)-2-(methoxycarbonyl)-6-(4-(4-vinylphenyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6) 
       
       
         
           
           
               
               
           
         
         g) heating a mixture of (2R, 3S, 4R, 5S, 6R)-2-(methoxycarbonyl)-6-(4-(4-vinylphenyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (6) as obtained in step (f), AIBN and a fourth solvent at a temperature in the range of 80° C. to 90° C. for a time period ranging from 24 to 25 hours to obtain a protected polymer (7) 
       
       
         
           
           
               
               
           
         
         h) adding sodium methoxide in a fifth solvent into the protected polymer (7) as obtained in step (g) in a solvent followed by stirring and refluxing for a time period ranging from 8 to 10 hours to obtain a polymer; and 
         i) dissolving the polymer as obtained in step (h), in a sixth solvent followed by adding hydrochloric acid and stirring at a temperature in the range of 25 to 30° C. for a time period ranging from 24 to 25 hours to obtain the water soluble polymer surfactant of  claim 1 . 
       
     
     
       3. The process according to  claim 2 , wherein the one or more of the first solvent, the second solvent, the third solvent, the fourth solvent, the fifth solvent, and the sixth solvent are selected from the group consisting of methanol, dichloromethane, tetrahydrofuran, dimethylformamide, water or a combination thereof. 
     
     
       4. A mini-emulsion polymerization process using a water soluble polymer surfactant, comprising the steps of:
 a) preparing an organic phase comprising a styrene, a co-stabilizer and a polymerizable (oligo) p-phenylenevinylene (OPV) dye; 
 b) preparing an aqueous phase comprising an initiator and the water soluble polymer surfactant of  claim 1  in a water; 
 c) adding the organic phase as obtained in step (a) drop-wise to the aqueous phase as obtained in step (b) and pre-emulsifying at a temperature in the range of 21° C. to 25° C. followed by sonication under ice cooled condition to obtain a mini-emulsion; 
 d) polymerizing the mini-emulsion as obtained in step (c) at a temperature of 70° C. for 20 hours with a stirring at a speed of 750 rpm to obtain a polymerized mini-emulsion; 
 e) quenching the polymerized mini-emulsion of step (d) to obtain a latex; and 
 f) purifying the latex as obtained in step (e), to obtain a glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead. 
 
     
     
       5. The process according to  claim 4 , wherein the quenching is achieved by adding two drops of 1 wt % hydroquinone. 
     
     
       6. The process according to  claim 4 , wherein the latex is purified by dialysis using a 6 kD MW cut-off membrane for three days. 
     
     
       7. The process according to  claim 4 , wherein the co-stabilizer is selected from the group consisting of hexadecane, cetyl alcohol, dodecyl methacrylate and stearyl methacrylate. 
     
     
       8. The process according to  claim 4 , wherein the initiator is selected from the group consisting of 4,4′-Azobis(4-cyanovaleric acid) (ACVA), azobisisobutyronitrile (AIBN), potassium peroxydisulfate (KPS), lactoperoxidase (LPO) and benzoyl peroxide (BPO). 
     
     
       9. The process according to  claim 4 , wherein the weight/weight (w/w) ratio of the water soluble polymer surfactant to (oligo) p-phenylenevinylene (OPV) dye is 1:3 to 5:3 in the glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead. 
     
     
       10. The process according to  claim 4 , wherein a size of the glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead is in the range of 163 to 328 nm. 
     
     
       11. A method of detection of bilirubin, comprising the steps of:
 a) preparing a sample solution in a water or a buffer at pH=10 by the addition of NaOH; 
 b) titrating the glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead as obtained in  claim 4  in a distilled water or a buffer with the sample solution of step (a); and 
 c) determining, by fluorimetry, a quenching of fluorescence intensity at 446 nm λmax, confirming the presence of bilirubin. 
 
     
     
       12. A kit for the detection of bilirubin, comprising:
 a) a stock solution of the glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead as obtained in  claim 4 ; 
 b) a graduated dropper; 
 c) an analysis chamber; and 
 d) a UV chamber. 
 
     
     
       13. A method of detection of bilirubin in a sample, comprising the steps of:
 a) adding a sample into a stock solution of the glucuronic acid functionalized polystyrene nanobead covalently incorporating (oligo) p-phenylenevinylene (OPV) nanosensor (PSG-OPV-n) bead as obtained in  claim 4  with the help of a dropper to obtain a sample solution; 
 b) pouring the sample solution of step (a) into an analysis chamber equipped with an UV chamber at the bottom; and 
 c) detecting a change in blue emission of the sample solution of step (b), confirming the presence of bilirubin in the sample. 
 
     
     
       14. The method according to  claim 13 , wherein the change in blue emission of the sample solution is visually detectable as a cyan color.